The objective of this proposal is to identify the specific heme pocket amino acids that convey peroxidase behavior upon CcP, and more generally to all peroxidases. This will be achieved by parallel kinetic and spectroscopic studies on rationally designed mutants of CcP. NMR will be used to monitor the pH behavior of H52, N82, R48 which are the most likely 'distal' heme pocket amino acids to function as primary reactivity regulators. Mutants of CcP derived from alterations at these positions will be screened by kinetics and their solution chemistry will be characterized by NMR. This will require expanding applications of NMR spectroscopy to paramagnetic proteins by identifying optimal protein size/field strength combinations for paramagnetic proteins and by expanding the repertoire of NMR applications for paramagnetic proteins to heteronuclei (13C and 15N). Uniformly isotope-labelled proteins necessary for this work will be created using the expression system for CcP that has been developed during the past grant period. Extensive protein engineering of CcP will occur. Its aim will be to create distal amino acid mutants of CcP that show altered reactivity towards H202 activation, thereby indicating which are critical for H202 activation. The primary method for screening these mutants will be kinetic measurement of the bimolecular rate constant for the initial reaction (in the CcP cycle) of Ccp and H202. Screening will be carried out in Professor James Erman's laboratory. A working hypothesis, based on limited published information, is that the CcP distal histidine (H52) is the primary regulator of heme ligand binding chemistry and H202 activation. We also propose to complete x-ray crystallographic structure determinations of the three major monomer hemoglobins (GMH2, GMH3, GMH4) from Glycera dibranchiata. None of these proteins have distal histidines; all have distal leucines and highly altered ligand binding dynamics and reactivity compared to Mb. For GMH4 we plan to create the Mb-mimic protein with a distal histidine: (LE7H)GMH4. Native and mutant will be kinetically studied to determine their bimolecular rate constants for reaction with H202. If our understanding of CcP bears out then we believe that we can enhance the rate of H202 activation in this protein via the PheB10His mutant, thereby taking the first step to engineer substantial non-natural enzymic activity into this ligand binding protein.